Nucleic acid enzymes for cleaving DNA

ABSTRACT

The present invention discloses nucleic acid enzymes capable of cleaving single-stranded DNA in a site specific manner.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation of copending U.S.application Ser. No. 08/007,732, filed Jan. 22, 1993, which is acontinuation of U.S. application Ser. No. 07/464,530, filed Jan. 12,1990 (abandoned), the disclosures of which are incorporated by referenceherein.

DESCRIPTION

[0002] This invention was made with government support under NASA GrantNo. NAGW-1671. The government may have certain rights in the invention.

TECHNICAL FIELD

[0003] The present invention relates to nucleic acid enzymes forcleaving DNA.

BACKGROUND

[0004] Some genes have their coding sequences interrupted by stretchesof non-coding DNA. These non-coding sequences are termed introns. Toproduce a mature transcript from these genes, the primary RNA transcript(precursor RNA) must undergo a cleavage-ligation reaction termed RNAsplicing. This RNA splicing produces the mature transcript of thepolypeptide coding messenger RNA (mRNA), ribosomal RNA, or transfer RNA(tRNA). Introns are grouped into four categories (Groups I, II, III, andIV) based on their structure and the type of splicing reaction theyundergo.

[0005] Of particular interest to the present invention are the Group Iintrons. Group I introns undergo an intra-molecular RNA splicingreaction leading to cyclization that does not require protein cofactors,Cech, Science, 236:1532-1539 (1987).

[0006] The Group I introns, including the intron isolated from the largeribosomal RNA precursor of Tetrahymena thermophila, have been shown tocatalyze a sequence-specific phosphoester transfer reaction involvingRNA substrates. Zaug and Cech, Science, 229:1060-1064 (1985); and Kayand Inoue, Nature, 327:343-346 (1987). This sequence-specificphosphoester transfer reaction leads to the removal of the Group Iintron from the precursor RNA and ligation of two exons in a processknown as RNA splicing. Splicing reaction catalyzed by Group I intronsproceeds via a two-step transesterification mechanism. The details ofthis reaction have been recently reviewed by Cech, Science,236:1532-1539 (1987).

[0007] The splicing reaction of Group I introns is initiated by thebinding of guanosine or a guanosine nucleotide to a site within theGroup I intron structure. Attack at the 5′ splice site by the3′-hydroxyl group of guanosine results in the covalent linkage ofguanosine to the 5′ end of the intervening intron sequence. Thisreaction generates a new 3′-hydroxyl group on the uridine at the 3′terminus of the 5′ exon. The 5′ exon subsequently attacks the 3′ splicesite, yielding spliced exons and the full-length linear form of theGroup I intron.

[0008] The linear Group I intron usually cyclizes following splicing.Cyclization occurs via a third transesterification reaction, involvingattack of the 3′-terminal guanosine at an interval site near the 5′ endof the intron. The Group I introns also undergo sequence specifichydrolysis reaction at the splice site sequences as described by Inoueet al., J. Mol Biol., 189:143-165 (1986). This activity has been used tocleave RNA substrates in a sequence specific manner by Zaug et al.,Nature, 324:429-433 (1986).

[0009] The structure of Group I introns has been recently reviewed by J.Burke, Gene, 73:273-294 (1988). The structure is characterized by ninebase paired regions, termed P1-P9 as described in Burke et al., NucleicAcids Res., 15:7217-7221 (1987). The folded structure of the intron isclearly important for the catalytic activity of the Group I introns asevidenced by the loss of catalytic activity under conditions where theintron is denatured. In addition, mutations that disrupt essentialbase-paired regions of the Group I introns result in a loss of catalyticactivity. Burke, Gene, 73:273-294 (1988). Compensatory mutations orsecond-site mutations that restore base-pairing in these regions alsorestore catalytic activity. Williamson et al., J. Biol. Chem.,262:14672-14682 (1987); and Burke, Gene, 73:273-294 (1988).

[0010] Several different deletions that remove a large nucleotidesegment from the Group I introns (FIG. 2) without destroying its abilityto cleave RNA have been reported. Burke, Gene, 73:273-294 (1988).However, attempts to combine large deletions have resulted in bothactive and inactive introns. Joyce et al., Nucleic Acid Res., 17:7879(1989).

[0011] To date, Group I introns have been shown to cleave substratescontaining either RNA, or RNA and DNA. Zaug et al., Science, 231:470-475(1986); Sugimoto et al., Nucleic Acids Res., 17:355-371 (1989); andCech, Science, 236:1532-1539 (1987). A DNA containing 5 deoxycytosineswas shown not to be a cleavage substrate for the Tetrahymena IVS, aGroup I intron by Zaug et al., Science, 231:470-475 (1986).

BRIEF SUMMARY OF THE INVENTION

[0012] It has now been discovered that Group I introns have the abilityto cleave single-stranded DNA substrates in a site specific manner.

[0013] Therefore the present invention provides a method of cleavingsingle-stranded DNA at the 3′-terminus of a predetermined nucleotidesequence present within single-stranded DNA. The single-stranded DNA istreated under DNA cleaving conditions with an effective amount of anendodeoxyribonuclease of the present invention where the DNA cleavingconditions include the presence of MgCl₂ at a concentration of at least20 millimolar.

[0014] The present invention also contemplates a composition containingan endodeoxyribonuclease enzyme of the present invention,single-stranded DNA and magnesium ion at a concentration of greater than20 millimolar.

[0015] The present invention further contemplates anendodeoxyribonuclease enzyme capable of cleaving single-stranded DNA ata predetermined nucleotide sequence where the enzyme has a nucleotidesequence defining a recognition site that is contiguous or adjacent tothe 5′-terminus of the nucleotide sequence, a first spacer regionlocated 3′-terminal to the recognition site, a P3 [5′] region located3′-terminal to the first spacer region, a second spacer region located3′-terminal to the P3 [5′] region, a first stem loop located 3′-terminalto the second spacer region, a second stem loop located 3′-terminal tothe first stem loop, a third spacer region located 3′-terminal to thesecond stem loop, and a third stem loop located 3′-terminal to the thirdspacer region, the third stem loop comprising a 5′ stem portioninterrupted by a nucleotide sequence defining a P3 [3′] region capableof hybridizing to the P3 [5′] region.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In the drawings forming a portion of this disclosure:

[0017] In FIG. 1, the splicing mechanisms of the four major groups ofprecursor RNAs. Wavy lines indicate introns, smooth lines indicateflanking exons. For nuclear mRNA splicing, many components assemble withthe pre-mRNA to form the spliceosome; only two, the U1 and U2 smallnuclear ribonucleoproteins, are shown. Nuclear tRNA splicing isdescribed by Greer et al., TIBS 9:139-41(1984).

[0018] In FIG. 2, the secondary structure of the T. thermophila pre-rRNAintron, with the recognition sequence and the core structure that is themost conserved region among group I introns shown in bold. Thenomenclature used to denote various structural features is the standardnomenclature described in Burke et al., Nucleic Acids Pes. 15:7217-7221(1987). The nine conserved pairing regions, P1-P9, and the various loopsare shown. The nucleotide sequence is numbered beginning at the 5′terminus of the molecule.

[0019] The recognition site is located at nucleotide 19 to 27, the firstspacer region is located at nucleotides 27 to 28 and 94 to 95, the P3[5′] region is located at nucleotides 96 to 103, the second spacerregion is located at nucleotides 104 to 106, the first stem loop islocated at nucleotides 107 to 214, the second stem loop is located atnucleotides 215 to 258, the third spacer region is located atnucleotides 259 to 261 and the third stem loop is located at nucleotides262 to 314.

[0020] In FIG. 3, the various deletions removing portions of the T.therophili pre-rRNA intron are shown. The nomenclature used is the samenomenclature defined in Burke et al., Nucleic Acids Research,15:7217-7221 (1987). The nucleotide segments removed in each deletionare shown with the greek character delta followed by the number of thepairing region removed. Combination of deletions are noted as Delta P2/9 for example.

[0021] In FIG. 4, trans-splicing activity of the wild-type and ΔP9mutant form of the Tetrahymena ribozyme using the substrateGGCCCUCU.A₃UA₃UA₃ (S1), d(GGCCCTCU.A₃TA₃TA) (S2), or d(GGCCCTCT.A₃TA₃TA)(S3) are shown.

[0022] In FIG. 5, selective amplification of the Tetrahymena ribozyme(E) based on its ability to react with an oligonucleotide substrate (S)is shown. Top, the L-21 form of the ribozyme binds anoligopyrimidine-containing RNA substrate by complementary pairing. The3′-terminal G_(OH) of the ribozyme attacks the phosphodiester bondfollowing a sequence of pyrimidines, resulting in transfer of the 3′portion of the substrate to the 3′ end of the ribozyme. Middle, theproduct of the RNA-catalyzed reaction offers a unique site forhybridization of an oligodeoxynucleotide used to initiate cDNAsynthesis. Bottom, a primer containing the T7 promoter is hybridized tothe cDNA, the second strand of the promoter is completed, the DNA istranscribed to RNA.

[0023] In FIG. 6, selective amplification of an ensemble of structuralvariants of the Tetrahymena ribozyme based on their ability to carry outa trans-splicing reaction with a DNA substrate. Lanes 1-3,trans-splicing with no substrate, the RNA substrate GGCCCUCU.A₃UA₃UA,and the DNA substrate d(GGCCCTCT.A₃TA₃TA). Lanes 4-5, selective cDNAsynthesis of trans-spliced products. Lanes 6-8, successive rounds oftranscription and reverse transcription leading to amplification ofselected materials. Materials were separated by electrophoresis in a 5%polyacrylamide/8M urea gel, an autoradiogram of which is shown.

DETAILED DESCRIPTION OF THE INVENTION

[0024] A. Enzymes

[0025] An endodeoxyribonuclease of the present invention is capable ofcleaving a single-stranded DNA substrate. Typically, theendodeoxyribonuclease is also capable of cleaving a single-stranded RNAsubstrate or a modified DNA substrate containing a uracil at thecleavage site rather than a thymine.

[0026] The term ribozyme is used to describe an RNA containing nucleicacid that is capable of functioning as an enzyme. Ribozymes includeendoribonucleases and endodeoxyribonucleases of the present invention.

[0027] An endodeoxyribonuclease of the present invention may be RNA,modified RNA, RNA-DNA polymer, a modified RNA-DNA polymer, a modifiedDNA-RNA polymer or a modified RNA-modified DNA polymer. RNA containsnucleotides containing a ribose sugar and adenine, guanine, uracil orcytosine as the base at the 1′ position. Modified RNA containsnucleotides containing a ribose sugar and adenine, thymine, guanine orcytosine and optionally uracil as the base. A RNA-DNA polymer containsnucleotides containing a ribose sugar and nucleotides containingdeoxyribose sugar and adenine, thymine and/or uracil, guanine orcytosine as the base attached to the 1′ carbon of the sugar. A modifiedRNA-DNA polymer is comprised of modified RNA, DNA and optionally RNA.Modified DNA contains nucleotides containing a deoxyribose sugar andnucleotides containing adenine, uracil, guanine, cytosine and possiblythymine as the base. A modified DNA-RNA polymer contains modified DNA,RNA and optionally DNA. A modified RNA-modified DNA polymer containsmodified RNA-modified DNA, and optionally RNA and DNA.

[0028] An endodeoxyribonuclease of the present invention is capable ofcleaving DNA 3′ of a predetermined base sequence. In addition, anendodeoxyribonuclease of this invention is characterized by a nucleotidesequence defining a recognition site that is contiguous or adjacent tothe 5′ terminus of the nucleotide sequence, a first spacer regionlocated 3′-terminal to the recognition site, a P3 [5′] region located3′-terminal to the IS first spacer region, a second spacer regionlocated 3′-terminal to the P3 [5′] region, a first stem loop located3′-terminal to the second spacer region, a second stem loop located3′-terminal to the first stem loop, a third spacer region located3′-terminal to the second stem loop, and a third stem loop located3′-terminal to the third spacer region, the third stem loop comprising a5′ stem portion defining a P3 [3′] region capable of hybridizing to theP3 [5′] region.

[0029] The recognition site of an endodeoxyribonuclease of the presentinvention contains a sequence of at least 2 to about 8 bases preferablyabout 4 to about 7 bases, capable of hybridizing to a complementarysequence of bases within the substrate nucleic acid giving theendodeoxyribonuclease its high sequence specificity. For example, anendodeoxyribonuclease of the present invention with a recognition sitebase sequence of 5′-GGAGG-3′ is able to recognize the base sequence5′-CCCTCT-3′ present within the single-stranded DNA substrate (seeExample 2). This same recognition site also allows theendodeoxyribonuclease to cleave modified DNA substrates with highsequence specificity (see Example 2.)

[0030] The exact bases present in the recognition site determine thebase sequence at which cleavage will take place. Cleavage of thesubstrate nucleic acid occurs immediately 3′ of the substrate cleavagesequence, the substrate nucleotide sequence that hybridizes to therecognition site. This cleavage leaves a 3′ hydroxyl group on thesubstrate cleavage sequence and a 5′ phosphate on the nucleotide thatwas originally immediately 3′ of the substrate cleavage sequence in theoriginal substrate. Cleavage can be redirected to a site of choice bychanging the bases present in the recognition sequence (internal guidesequence). See Murphy et al., Proc. Natl. Acad. Sci., USA, 86:9218-9222(1989). (The disclosures of all references cited within this documentare incorporated by reference.) In addition, any combination of basesmay be present in the recognition site if a polyamine is present. See,for example, Doudna et al., Nature, 339:519-522 (1989). Typically, thepolyamine is either spermidine, putrescine or spermine. A spermidineconcentration of about 5 mM was shown to be effective. The recognitionsite may also be provided as a separate nucleic acid, an externalrecognition site not covalently coupled to the rest of theendodeoxynuclease. External recognition sites have been shown to directendoribonuclease cleavage at a specific base sequence by Doudna et al.,Nature, 339:519-522 (1989). If an external recognition site is used, theendodeoxyribonuclease used with it would not contain a recognition sitebut would comprise a P3 [5′] region, a second spacer region, a firststem loop, a second stem loop, a third spacer region and a third stemloop where the third stem loop comprises a 5′ stem portion defining a P3[3′] region capable of hybridizing to said P3 [5′] region.

[0031] Use of an endodeoxyribonuclease of the present invention with anexternal recognition site would allows the target sequence to be changedby merely changing the external recognition site sequence. Use of aplurality of different external recognition sequences with anendodeoxyribonuclease of the present invention allows the substratenucleic acid to be cleaved at each of the different base sequencesencoded by the external recognition sequences.

[0032] First spacer regions typically contain a sequence of nucleotidesabout 3 bases to about 7 bases, preferably about 5, bases in length.Preferably, the nucleotides making up the first spacer have the sequence5′-NNNNA-3′, where N represents the presence of any nucleotide at thatposition. More preferably, the first spacer region is defined by thesequence 5′-AACAA-3′.

[0033] In other preferred embodiments, the first spacer region iscomprised of a nucleotide sequence defining two spacer stem loops.Preferably, the first spacer stem loop is 25 nucleotides in length, andthe second spacer stem loop is 36 bases in length. More preferably, thefirst spacer stem loop has the base sequence,5′-AGUUACCAGGCAUGCACCUGGUAGUCA-3′, and the second spacer stem loop hasthe base sequence, 5′-GUCUUUAAACCAAUAGAUU-GGAUCGGUUUAAAAGGC-3′.

[0034] A stem loop is a secondary structure formed by a nucleotidesequence that has “folded over on itself”. A stem loop comprises a 5′nucleotide sequence portion, designated a 5′ paring segment (P[5′]) thatis capable of hybridizing to a nucleotide sequence located 3′ of theP[5′] and is designated the 3′ pairing segment (P[3′]). In a stem loop,the P[5′] and P[3′] are connected by a nucleotide sequence called aloop. The P[5′] and P[3′] hybridize and form a nucleic acid duplex. Thenucleic acid duplex formed by the P[5′] and P[3′] does not have to be aperfect duplex and may contain stretches of nucleotides that are eitherunpaired or paired to a sequence outside the stem loop.

[0035] In preferred embodiments, the P3 [5′] region is an eightnucleotide sequence. The eight nucleotides present in the P3 [5′] regionmay be any eight nucleotides as long as the P3 [5′] region is capable ofhybridizing with the P3 [3′] region to form the third pairing segment orP3 shown in FIG. 2. The formation of P3 by P3 [5′] region and P3 [3′]region are required for catalytic activity as has been recently reviewedby John Burke, Gene, 73:273-294 (1988).

[0036] More preferably, the P3 [5′] has the nucleotide sequence,5′-GACCGUCA-3′. However, changes in the P3 [5′] region nucleotidesequences may be made as long as P3 is still able to form or ifcompensating changes in the P3 [3′] region nucleotide sequence have beenmade as has been demonstrated by Williamson et al., J. Biol. Chem.,262:14672-14682 (1987).

[0037] In preferred embodiments, the second spacer region is about threenucleotides in length. Typically, any three nucleotides may make up thesecond space region as long as the ribozyme containing this spacer hasthe desired catalytic activity.

[0038] More preferably, the second spacer region has the nucleotidesequence, 5′-AAU-3′. However, this sequence may be changed as long asthe desired catalytic activity is retained.

[0039] In preferred embodiments, the first stem loop is about 108nucleotides in length. More preferred, the first stem loop correspondsto nucleotides 107 to 214 of FIG. 2. The ten most 5′-terminalnucleotides (nucleotides 107 to 117 of FIG. 2) and the ten most3′-terminal nucleotides (nucleotides 204 to 214 of FIG. 2) of the firststem loop are the most critical for known catalytic functions. See, forexample, John Burke, Gene, 73:273-294 (1988).

[0040] In other preferred embodiments, the first stem loop is about 39nuclectides in length. More preferred, the first stem loop correspondsto nucleotides 107 to 126 and nucleotides 196 to 214 in FIG. 2, wherenucleotide 126 is directly linked to nucleotide 196.

[0041] In another preferred embodiment, the first stem loop is 20nucleotides in length. More preferred, the first stem loop correspondsto nucleotides 107 to 116, and nucleotides 205 to 214 of FIG. 2, wherenucleotide 116 is directly linked to nucleotide 205.

[0042] In preferred embodiments, the second stem loop is about 44nucleotides in length. More preferred, the first stem loop correspondsto nucleotides 215 to 258 in FIG. 2. The 5 most 5′-terminal nucleotides(nucleotides 215 to 219 in FIG. 2) and the 5 most 3′-terminalnucleotides (nucleotides 254 to 258 in FIG. 2) of the second stem loopare the most critical For known catalytic functions. See for example,John Burke, Gene, 73:273-294 (1988).

[0043] In other preferred embodiments, the second stem loop is about 25nucleotides in length. More preferably, the second stem loop nucleotidesequence substantially corresponds to nucleotides 215 to 227 andnucleotides 247 to 258 in FIG. 2, where nucleotides 227 and 247 aredirectly linked. In another preferred embodiment, the second stem loopis about 9 nucleotides in length. More preferably, the second stem loopsubstantially corresponds to nucleotides 215 to 220 and 256 to 258 inFIG. 2 where nucleotides 220 and 256 are directly linked.

[0044] In preferred embodiments, the third spacer region is about 3nucleotides in length. More preferably, the third spacer regioncorresponds to nucleotides 259 to 261 of FIG. 2. Some nucleotide changescan be made in the third spacer region while still preserving thedesired catalytic activity. See for example, Williamson et al., J. Biol.Chem., 262:14672-14682 (1987), where the nucleotide at position 259 waschanged to A and the nucleotide at 261 was changed to a C whilemaintaining splicing activity. The nucleotide at position 260 (FIG. 2)was changed to either G, A or U while preserving the desired catalyticactivity as reported in John Burke, Gene, 73:273-294 (1988).

[0045] In preferred embodiments, the third stem loop is about 51nucleotides in length. These 51 nucleotides are divided into a 5′ stemportion defining a P3 [3′] region that is capable of hybridizing to theP3 [5′] region, a loop and a 3′ stem portion. Preferably, the P3 [3′]region is about 8 nucleotides in length. However, the length of the P3[3′] may vary to correspond with the length of the P3 [5′] region.Preferably, the P3 [3′] region begins about 10 nucleotides from the 5′end of the third stem loop.

[0046] More preferably, the third stem loop is 5 nucleotides in lengthand those nucleotides substantially correspond to nucleotides 262 to 312in FIG. 2. Preferably, the P3 [3′] region is about 8 nucleotides inlength and those nucleotides substantially correspond to nucleotides 271to 278 in FIG. 2. Changes, including deletions, mutations, reversionsand insertions, can be made within the third stem loop and the P3 [3′]region and still maintain the desired catalytic activity. See, forexample, Burke et al., Cell, 45:167-176 (1986) and Williamson et al., J.Biol. Chem., 262:14672-14682 (1987), where nucleotide 266 was changed toG and a compensatory mutation changing nucleotide 309 to C was madewhile maintaining the desired catalytic activity. Other mutations,including changing nucleotide 268 to C and at the same time changingnucleotide 307 to C, and changing nucleotide 268 to U and at the sametime changing nucleotide 307 to A, were also shown to maintain thedesired catalytic activity.

[0047] Other changes in the nucleotide sequence of the third stem loopare also contemplated by the present invention. Changing nucleotides 301to C (FIG. 2), 302 to 6, and 303 to C has been shown to eliminatetransesterification activity, but does not eliminate site specificcleavage or GTP binding by Williamson et al., J. Biol. Chem.,262:14672-14682 (1987).

[0048] Charges in nucleotides 280 and 282 (FIG. 2) together withcompensatory changes in nucleotides 296 and 298 have been shown topreserve the desired catalytic function as reported in J. Burke, Gene,73:273-294 (1988). Mutations such as these preserve a given secondarystructure and these and similar mutations would be expected to maintainthe desired catalytic activity.

[0049] Changes in the P3 [3′] region, nucleotides 272 and 274 (FIG. 2),along with compensatory changes in nucleotides 100 and 102, were madewhile maintaining the desired catalytic activity by Williamson et al.,J. Biol. Chem., 262:14672-14682 (1987) and Inoue et al., Cell,43:431-437 (1985). Changes similar to those changes made above willmaintain the desired catalytic activity as long as the particularsecondary structure such as a stem loop, pairing region or spacer ismaintained.

[0050] An endodeoxyribonuclease of the present invention may alsoinclude additional stem loops located 3′-terminal to the third stemloop. These additional stem loops may contain any number of stem loopsas long as the desired catalytic activity is maintained. Preferably, anyadditional stem loops have a nucleotide sequence that substantiallycorresponds to nucleotides 316 to 402 of FIG. 2.

[0051] In preferred embodiments, an endodeoxyribonuclease of the presentinvention may combine one or more of the mutations described above.Typically, these deletions change the length of or alter the nucleotidesequence of a stem loop, the P3 [5′], the P3 [3′] region, a spacerregion or the recognition sequence. The mutation within onecatalytically active endodeoxyribonuclease may be combined with themutation within a second catalytically active endodeoxyribonuclease toproduce a new endodeoxyribonuclease containing both mutations.

[0052] In other preferred embodiments, an endodeoxyribonuclease of thepresent invention may have random or defined mutations introduced intoit using a variety of methods well known to those skilled in the art.For example, the method described by Joyce et al., Nucleic AcidsResearch, 17:711-712 (1989), involves excision of template (coding)strand of double-stranded DNA, reconstruction of the template strandwith inclusion of mutagenic oligonucleotides, and subsequenttranscription of the partially-mismatched template. This allows theintroduction of defined or random mutations at any position in themolecule by including polynucleotides containing known or randomnucleotide sequences at selected positions. Alternatively, mutations maybe introduced into the endodeoxyribonuclease by substituting 5-Br dUTPfor TTP in the reverse transcription reaction. 5-Br dU can pair with dGin the “wobble” position as well as dA in the standard Watson-Crickposition, leading to A to G and G to A transitions. Similarly,substituting 5-Br UTP for UTP in the forward transcription reactionwould lead to C to U and U to C transitions in the subsequent found ofRNA synthesis.

[0053] B. Methods

[0054] The method of the present invention is useful for cleaving anysingle-stranded nucleic acid including single-stranded DNA, modifiedDNA, RNA and modified RNA. The single-stranded nucleic acid must only besingle-stranded at or near the substrate cleavage sequence so that anenzyme of the present invention can hybridize to the substrate cleavagesequence by virtue of its recognition sequence.

[0055] A single-stranded nucleic acid that will be cleaved by a methodof this invention may be chemically synthesized, enzymatically producedor isolated from various sources such as phages, viruses or cells,including plant cells, eukaryotic cells, yeast cells and bacterialcells. Chemically synthesized single-stranded nucleic acids arecommercially available from many sources including, Research Genetics,Huntsville, Alabama. Single-stranded phages such as the M13 cloningvectors described by Messing et al., Proc. Natl. Acad. Sci., USA,74:3642-3646 (1977), and Yanisch-Perron et al., Gene, 33:103-119 (1985).Bacterial cells containing single-stranded phages would also be a readysource of suitable single-stranded DNA. Viruses that are eithersingle-stranded DNA viruses such as the parvoviruses or are partiallysingle-stranded DNA viruses such as the hepatitis virus would providesingle-stranded DNA that could be cleaved by a method of the presentinvention. Single-stranded RNA cleavable by a method of the presentinvention could be provided by any of the RNA viruses such as thepicornaviruses, togaviruses, orthomyxoviruses, paramyxoviruses,rhabdoviruses, coronaviruses, arenaviruses or retroviruses.

[0056] The methods of this invention may be used on single-strandednucleic acid that are present inside a cell, including eucaryotic,procaryotic, plant, mammalian, yeast or bacterial cell. Under theseconditions a method of the present invention could act as an anti-viralagent or a regulatory of gene expression.

[0057] The method of the present invention cleaves single-stranded DNAat the 3′-terminus of a predetermined base sequence. This predeterminedbase sequence or substrate cleavage sequence may contain from 2 to 8nucleotides. The method allows cleavage at any nucleotide sequence byaltering the nucleotide sequence of the recognition site of theendodeoxyribonuclease. This allows cleavage of single-stranded DNA inthe absence of a restriction endonuclease site at that position.

[0058] Cleavage at the 3′-terminus of a predetermined base sequenceproduces a single-stranded DNA, containing the substrate cleavagesequence, with a 3′-terminal hydroxyl group. In addition, the cleavagejoins the remainder of the original single-stranded DNA substrate withthe endodeoxyribonuclease enzyme. This cleavage reaction and productsproduced from this cleavage reaction are analogous to the cleavagereaction and cleavage products produced by the Tetrahymena ribozymedescribed by Zaug and Cech, Science, 231:470-475 (1986) and reviewed byT. R. Cech, Annual Rev. of Biochem., 59:(1990). Theendodeoxyribonuclease of the present invention may be separated from theremainder of single-stranded DNA substrate by site-specific hydrolysisat the phosphodiester bond following the 3′-terminal guanosine of theendodeoxyribonuclease similar to the site-specific cleavage at thisposition described for the ribozyme acting on RNA by Inoue et al., J.Mol. Biol., 189:143-165 (1986). Separation of the endodeoxyribonucleasefrom the substrate allows the endodeoxyribonuclease to carry out anothercleavage reaction.

[0059] Single-stranded DNA is treated under DNA cleaving conditions withan effective amount of an endodeoxyribonuclease of the presentinvention, where the DNA cleaving conditions include the presence ofMgCl₂ at a concentration of at least 20 millimolar.

[0060] An effective amount of an endodeoxyribonuclease is the amountendodeoxyribonuclease required to cleave a predetermined base sequencepresent within the single-stranded DNA. Preferably, theendodeoxyribonuclease is present at a molar ratio ofendodeoxyribonuclease to substrate cleavage sites of 1 to 20. This ratiomay vary depending on the length of treating and efficiency of theparticular endodeoxyribonuclease under the particular DNA cleavageconditions employed.

[0061] Treating typically involves admixing, in aqueous solution, thesingle-stranded DNA, the enzyme and the MgCl₂ to form a DNA cleavageadmixture, and then maintaining the admixture thus formed under DNAcleaving conditions for a time period sufficient for theendodeoxyribonuclease to cleave the single-stranded DNA at any of thepredetermined nucleotide sequences present in the single-stranded DNA.

[0062] Preferably, the amount of time necessary for theendodeoxyribonuclease to cleave the single-stranded DNA has beenpredetermined. The amount of time is from about 5 minutes to about 24hours and will vary depending upon the concentration of the reactants,and the temperature of the reaction. Usually, this time period is fromabout 30 minutes to about 4 hours such that the endodeoxyribonucleasecleaves the single-stranded DNA at any of the predetermined nucleotidesequences present.

[0063] Preferably, the DNA cleaving conditions include the presence ofMgCl₂ at a concentration of at least 20 mM. Typically, the DNA cleavingconditions include MgCl₂ at a concentration of about 20 mM to about 150mM. The optimal MgCl₂ concentration to include in the DNA cleavingconditions can be easily determined by determining the amount ofsingle-stranded DNA cleaved at a given MgCl₂ concentration. One skilledin the art will understand that the optimal MgCl₂ concentration may varydepending on the particular endodeoxyribonuclease employed.

[0064] Preferably, the DNA cleaving conditions are at from about pH 6.0to about pH 9.0. One skilled in the art will understand that the methodof the present invention will work over a wide pH range so long as thepH used for DNA cleaving is such that the endodeoxyribonuclease is ableto remain in an active conformation. An endodeoxyribonuclease in anactive conformation is easily detected by its ability to cleavesingle-stranded DNA at a predetermined nucleotide sequence. Morepreferably, the DNA cleaving conditions are at from about pH 7.0 toabout pH 8.0. Most preferred are DNA cleaving conditions at about pH7.5.

[0065] Preferably, the DNA cleaving conditions are at from about 15° C.to about 60° C. More preferably, the DNA cleaving conditions are fromabout 30° C. to about 56° C. The temperature of the DNA cleavingconditions are constrained only by the desired cleavage rate and thestability of that particular endodeoxyribonuclease at that particulartemperature. Most preferred are DNA cleavage conditions from about 37°C. to about 50° C.

[0066] In other preferred methods the present invention contemplates DNAcleaving conditions including the presence of a polyamine. Polyaminesuseful practicing the present invention include spermidine, putrescine,spermine and the like. Preferably, the polyamine is spermidine and it ispresent at a concentration of about 1 mM to about 15 mM. Morepreferably, spermidine is present at a concentration of about 1 mM toabout 10 mM. Most preferred, are DNA cleavage conditions including thepresence of spermidine at a concentration of about 2 mM to about 5 mM.

[0067] The present invention also contemplates a method of producing anucleic acid having a predetermined activity. Preferably, the desiredactivity is a catalytic activity.

[0068] A population of Group I nucleic acids is subjected tomutagenizing conditions to produce a diverse population of mutantnucleic acids.

[0069] In preferred embodiments, the population of Group I nucleic acidsis made up of at least 2 Group I nucleic acids. Group I nucleic acidsare nucleic acid molecules having at least a nucleic acid sequencedefining a recognition site that is contiguous or adjacent to the5′-terminus of the nucleotide sequence, a first spacer region located3′-terminal to the recognition site, a P3 [5′] region located3′-terminal to the first spacer region, a second spacer region located3′-terminal to the P3 [5′] region, a first stem loop located 3′-terminalto the second spacer region, a second stem loop located 3′-terminal tothe first stem loop, a third spacer region located 3′-terminal to thesecond stem loop, and a third stem loop located 3′-terminal to the thirdspacer region, the third stem loop comprising a 5′ stem portion defininga P3 [3′] region capable of hybridizing to the P3 [5′] region.

[0070] In preferred embodiments, mutagenizing conditions includeconditions that introduce either defined or random nucleotidesubstitutions within the Group I nucleic acid. Examples of typicalmutagenizing conditions include conditions disclosed in other parts ofthis specification and the methods described by Joyce et al., NAR17:711-722(1989) and Joyce, Gene, 82:83-87(1989).

[0071] In preferred embodiments, a diverse population of mutant nucleicacid contains at least 2 nucleic acid molecules that do not have theexact same nucleotide sequence.

[0072] A nucleic acid having a predetermined activity is selected fromthe diverse population of mutant nucleic acids on the basis of itsability to perform the predetermined activity.

[0073] In preferred embodiments, selecting includes any means ofphysically separating the mutant nucleic acids having a predeterminedactivity from the diverse population of mutant nucleic acids. Typically,selecting includes separation by size, the presence of a catalyticactivity and hybridizing the mutant nucleic acid to another nucleic acidthat is either in solution or attached to a solid matrix. Preferably,the predetermined activity is such that the mutant nucleic activityhaving the predetermined activity becomes labelled in some fashion byvirtue of the activity. For example, the predetermined activity may bean endodeoxyribonuclease activity whereby the activity of the mutantnucleic acid upon its substrate causes the mutant nucleic acid to becomecovalently linked to it. The mutant nucleic acid is then selected byvirtue of the covalent linkage.

[0074] In other preferred embodiment, selecting a mutant nucleic acidhaving a predetermined activity includes amplification of the mutantnucleic acid as described in Joyce, Gene, 82:83-87(1989).

[0075] D. Compositions

[0076] Also contemplated by the present invention are compositionscontaining an endodeoxyribonuclease enzyme of the present invention,single-stranded DNA and magnesium ion at a concentration of greater than20 millimolar.

[0077] Preferably, the endodeoxyribonuclease is present at aconcentration of about 0.05 μM to about 2 mM. Typically, theendodeoxyribonuclease is present at concentration ration ofendodeoxyribonuclease to single-stranded DNA from about 1 to 5 to about1 to 50. More preferably, the endodeoxyribonuclease is present in thecomposition at a concentration of about 0.1 μM to about 1 μM. Mostpreferred, are compositions containing the endodeoxyribonuclease at aconcentration of about 0.1 μM to about 0.5 μM.

[0078] Preferably, single-stranded DNA is present in the composition ata concentration of about 0.5 μM to about 1000 μM. One skilled in the artwill understand that there are many sources of single-stranded DNAincluding synthetic DNA, phage DNA, denatured double-stranded DNA, viralDNA and cellular.

[0079] Preferably, magnesium ion is present in the composition at aconcentration of about 20 mM to about 200 mM. More preferably, themagnesium ion is present in the composition at a concentration of about20 mM to about 150 mM. One skilled in the art will understand that themagnesium ion concentration is only constrained by the limits ofsolubility of magnesium in aqueous solution and a desire to have theendodeoxyribonuclease present in the same composition in an activeconformation.

[0080] Also contemplated by the present invention are compositionscontaining an endodeoxyribonuclease enzyme of the present invention,single-stranded DNA, magnesium ion at a concentration of greater than 20millimolar and a polyamine.

[0081] Preferably, the polyamine is spermidine, putrescine, or spermine.More preferably, the polyamine is spermidine and is present at aconcentration of about 2 mM to about 10 mM.

[0082] Also contemplated by the present invention are compositioncontaining an endodeoxyribonuclease enzyme of the present invention,single-stranded DNA, magnesium ion at a concentration of greater than 20millimolar, a second single-stranded DNA molecule ending in a3′-terminal hydroxyl, and a third single-stranded DNA molecule having aguanine nucleotide at its 5′-terminal end.

[0083] Also contemplated by the present invention are compositionscontaining an endodeoxyribonuclease enzyme of the present invention,singled-stranded DNA and magnesium ion at a concentration of greaterthan 20 millimolar, wherein said single-stranded DNA is greater inlength than the recognition site present on the endodeoxyribonucleaseenzyme.

EXAMPLES

[0084] The following examples illustrate, but do not limit, the presentinvention.

[0085] 1. Preparation of Endodeoxyribonucleases.

[0086] The wild-type and mutant ribozymes were produced by firstisolating the 443 base-pair Eco RI to Hind III restriction endonucleasefragment from the plasmid PT7-21 described by Zaug et al., Biochemistry,27:8924 (1988) using the standard methods described in Current Protocolsin Molecular Biology, Ausubel et al., eds. John Wiley and Sons, New York(1987).

[0087] This 443 base-pair fragment contains the T7 promoter described byDunn et al., J. Mol. Biol., 166:477-535 (1983) and residues 22-414 ofthe Tetrahymena IVS and residues 1-25 of the 3′ Tetrahymena exondescribed by Been et al., Cell, 47:207-216 (1986). This Eco RI and HindIII fragment was inserted into the M13 vector, M13mp18 that is similarto the vector described by Yanisch-Perron et al., Gene, 33:103-119(1985), that had been previously cleaved with Eco RI Hind III, and usingstandard subcloning procedures described in Current Protocols inMolecular Biology, Ausubel et al, eds. John Wiley and Sons, New York(1987). The resulting M13T7L-21 DNA construct was transformed into E.coli host cells according to the transformation procedure described inMolecular Cloning: A Laboratory Manual, Maniatis et al., eds., ColdSpring Harbor Laboratories, Cold Spring Harbor, New York (1989). Singlestranded DNA was then prepared from the M13T7L-21 transformed cellsaccording to the procedures described in Current Protocols in MolecularBiology, Ausubel et al, eds. John Wiley and Sons, New York (1987). Theaccuracy of the above construction was confirmed by DNA sequencing usingthe klenow fragment of E. coli DNA polymerase I (Boehringer MannheimBiochemicals, Indianapolis, Ind.) and the dideoxynucleotide sequencingmethod described by Sanger et al., Proc. Natl. Acad. Sci., USA,74:5463-5467 (1977).

[0088] The wild-type and mutant ribozymes were prepared directly fromthe single-stranded M13T7L-21 DNA using a modification of the techniquepreviously described by Joyce and Inoue, Nucleic Acid Research,17:711-722 (1989). The technique involves construction of a templatestrand that optionally includes one or more mutagenicoligodeoxynucleotides. The resulting partially-mismatcheddouble-stranded DNA is transcribed directly using T7 RNA polymerase.Briefly, a five fold molar excess of a terminator polynucleotide and amutator oligo were admixed with 5 μg of single-stranded M13T7L-21 DNAand a solution containing 20 mM tris[hydroxy-methyl]aminomethaneadjusted to pH 7.5 with HCl(Tris-HCl), 50 mM NaCl and 2 mM MgCl₂. Thissolution was maintained at 70 degrees centigrade (70° C.) for 5 minutesand then steadily cooled to 30° C. over 40 minutes. Fifteen units(U) ofT4 DNA ligase (U.S. Biochemicals, Cleveland, Ohio) and 7.5 U of T4 DNApolymerase (U.S. Biochemicals) were admixed to the solution togetherwith sufficient amounts of reagents to make the solution contain a finalconcentration of 20 mM Tris-HCl at pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 2 mMdithiothreitol (DTT), 1 mM adenosine triphosphate (ATP) and 0.5 mM eachof dGTP, dTTP, dATP and dCTP (dNTPs). The resulting solution wasmaintained at 37° C. for 60 minutes to complete the synthesis of themutant strand. The resulting DNA was purified by ethanol precipitationand then used to direct the transcription of mutant RNA.

[0089] Transcription took place either in a 10 μl volume containing 1 μgof mutant DNA, 2 μCi [α³²P] GTP and 50 U of T7 RNA polymerase that wasprepared as previously described by Davanloo et al., Proc. Natl. Acad.Sci., USA, 81:2035-2039 (1984), and purified according to a procedureoriginally developed by Butler & Chamberlain, J. Bio. Chem.,257:5772-5779 (1982), or in a 400 μl volume containing 10 μg of mutantDNA, 40 μCi [³H] UTP and 2,400 U of T7 RNA polymerase. In either case,the transcription mixture also contained 40 mM Tris-HCl at pH 7.5, 15 mMMgCl₂, 10 mM dithiothreitol, 2 mM spermidine, and 1 mM (each) NTPS, andwas incubated at 37° C. for 90 minutes. The T7 RNA polymerase wasextracted with phenol and the transcription products were purified byethanol precipitation. The mutant RNA was isolated by electrophoresis ina 5% polyacrylamide/8 M urea gel, eluted from the gel, and purified byethanol precipitation and chromatography on Sephadex G-50.

[0090] The Delta P2 mutant was prepared using the mutagenicoligodeoxynucleotide 01 (Table 1 and FIG. 3). The partially-randomizedDelta P2 mutant was prepared using the mutagenic oligodeoxynucleotide 02(Table 1 and FIG. 3). The Delta PS mutant was prepared using mutagenicoligonucleotides 03 or 04 (Table 1 and FIG. 3). The Delta P6 mutant wasprepared using the mutagenic oligodeoxynucleotide 05 (Table 1 and FIG.3). The Delta P6b mutant was prepared using the mutagenicoligodeoxy-nucleotide 06 (Table 1 and FIG. 3). The Delta P9 mutant wasprepared using the mutagenic oligodeoxy-nucleotide 07 (Table 1 and FIG.3).

[0091] Wild-type and mutant RNAs other than those containing the DeltaP9 deletion were defined at their 3′ end by the oligodeoxynucleotide 08(Table 1 and FIG. 3). Mutants containing the Delta P9 deletion weredefined by the Delta P9 mutagenic oligo which directs a transcript thatincludes 10 nucleotides of the 3′ exon. TABLE 1 01)5′-TTTGACGGTCTTGTTCCCTCCTATAGTGAG-3′ 02)5′-TTTGACGGTCTNNNNCCCTCCTATAGTGAG-3′ 03) 5′-TGCGTGGTTACTTTCCCGCAA-3′ 04)5′-GGACTTGGCTGCGTGGTTACTTTCCCGCAA-3′ 05) 5′-TTTAGTCTGTGAACTCTTGGC-3 06)5′-TCTGTGAACTGCATCCAAGCTTAGGACTTGG-3′ 07)5′-GGCTACCTTACGAGTACTCCGACTATATCTTAT-3′ 08) 5′-CGAGTACTCCAAAAC-3′

[0092] The 3′ exon sequence was removed by RNA-catalyzed site-specifichydrolysis as has been previously, Inoue et al., J. Mol. Biol.,189:143-165 (1986). Briefly, the RNA was incubated in the presence of 50mM CHES at pH 9.0 and 10 mM MgCl₂ at 42° C. for 1 hour. Wild-type andmutant RNAs were isolated by electrophoresis in a 5% polyacrylamide/8Murea gel, eluted from the gel, and purified by affinity chromatographyon du Pont Nensorb (du Pont Company, Wilmington, Del.). RNAs weresequenced by primer extension analysis using AMV reverse transcriptase(Life Technologies, Inc., Gaithersburg, Md.) in the presence ofdideoxynucleotides, using a modification of the methods described bySanger et al., Proc. Natl. Acad. Sci., USA, 74:5463-5467 (1977), exceptfor those containing the Delta P9 deletion, which were sequenced fromthe 3′ end by partial RNase digestion, Donis-Keller et al., NucleicAcids Res., 15:8783-8798 (1987).

[0093] The RNA substrate 5′-GGCCCUCUA₁₃-3′ was prepared by in vitrotranscription using a partially single-stranded synthetic DNA templateaccording to the methods described by Milligan et al., Nucleic AcidsRes., 4:2527-2538 (1977). The template contains both strands of thepromoter for T7 RNA polymerase (positions −17 through +1) followed bythe single-stranded template sequence 3′-CGGGAG- AT₁₀-5′. Run-offtranscripts of the form 5′-GGCCCUCUA_(n)-3′, where n=9-16, wereobtained. The resulting products were separated by electrophoresis in a20% polyacrylamide/8M urea gel, eluted from the gel, purified byaffinity chromatography on du Pont Nensorb, and sequenced by partialRNase digestion Donis-Keller et al., Nucleic Acids Research,15:8783-8798 (1987). RNA substrates having the sequence5′-GGCCCUCUA₁₃-3′ were used throughout this study.

[0094] The DNA substrates were either purchased from a number ofcommercial sources (i.e., Research Genetics, Huntsville, Ala.) orsynthesized using an Applied Biosystems (Foster City, Calif.)oligonucleotide synthesizer according to the manufacture's instructions.

[0095] 2. Cleavage of single-stranded DNA by Endodeoxyribonuclease

[0096] The ability of the Delta P9 mutant and wild-type ribozymes tocleave three different substrates was determined. The reactions werecarried out by admixing 0.02M of the ribozyme, 2.0 μM of eitherGGCCCUCU.A₃UA₃UA₃ (S1) or d(GGCCCTCU.A₃TA₃TA) (S2) ord(GGCCCTCT.A₃TA₃TA) (S3), 30 mMN-[2-hydroxyethyl]-piperazine-N′-[3-propane-sulfonic acid] (EPPs) at pH7.5, 50 mM MgCl₂ and 2 mM spermidine. The resulting solution wasmaintained at 50° C. for one hour. The resulting reaction products wereseparated by electrophoresis in a 5% polyacrylamide/8m urea gel. The gelwas used to expose x-ray film to produce an autoradiogram shown in FIG.4.

[0097] The Delta P9 ribozyme cleaves the RNA substrate, S1 the modifiedDNA substrate, S2, and the DNA substrate S3 (FIG. 4).

[0098] 3. Selection of Mutant Ribozymes Capable of Cleaving DNA.

[0099] Mutant Ribozymes capable of cleaving a DNA substrate wereselected using the in vitro evolution system described by G. F. Joyce,Gene, 82:83-87 (1989). This technique allows a structural variant ofTetrahymena ribozyme capable of catalyzing a specific reaction to beselectively amplified from a population of Tetrahymena ribozymestructural variants.

[0100] This in vitro evolution technique was used to select aTetrahymena ribozyme structural variant that cleaves apolydeoxyribonucleic acid (FIG. 5). The first step in this technique isthe ribozyme trans-splicing reaction involving the attack by its3′-terminal guanosine at a phosphodiester bond following a sequence ofpyrimidines located within a RNA substrate previously described by G. F.Joyce, Gene, 82:83-87 (1989). The product of the reaction is theribozyme joined to the substrate sequence that lies downstream from thetarget phosphodiester (FIG. 5, top). Selection occurs when anoligodeoxynucleotide primer is hybridized across the ligation junctionand used to initiate synthesis of complementary using reversetranscriptase DNA (FIG. 5, bottom). The primer ligation junction doesnot bind to unreacted starting materials (<10⁻⁶ compared to reactionproducts, at or below the limits of detection), and thus leads toselective reverse transcription of reactive materials. In order toamplify the selected materials, a primer containing one strand of apromoter for T7 RNA polymerase is hybridized to the extreme 3′ end ofthe cDNA, the second strand of the promoter is completed using aDNA-dependent DNA polymerase, and the DNA is transcribed to RNA as hasbeen previously described by Joyce, G. F. in Molecular Biology of RNAUCLA Symposia on Molecular and Cellular Biology, ed., Cech, T. R.,94:361-371, Alan R. Liss, New York, (1989) and Kwoh et al., Proc. Natl.Acad. Sci., USA, 86, 1173-1177 (1989).

[0101] The selected material is amplified at the transcription level dueto the high turnover of T7 RNA polymerase that has been previouslydescribed by Chamberlin et al., in The Enzymes, ed. P. Boyer, pp.87-108, Academic Press, New York (1982). Mutations can be introduced byreplacing a portion of the cDNA with one or more mutagenicoligodeoxynucleotides and transcribing the partially-mismatched templatedirectly as has been previously described by Joyce et al., Nucleic AcidResearch, 17:711-722 (1989). Ribozymes produced in this way can also beinternally labelled with ³²P-GTP.

[0102] The ability of a population of wild-type and mutant forms of theTetrahymena ribozyme to cleave the RNA substrate GGCCCUCUAAAUAAAUA (S1),the modified DNA substrate d(GGCCCTCUAAATAAATA) (S2), and the DNAsubstrate d(GGCCCTCTAAATAAATA) (S3) was determined. Briefly, 1 μM eachof internally labelled wild-type, Delta P6, Delta P2, Delta P9, DeltaP6/P9 and Delta P2/P9 ribozymes were admixed with 2 μM of either the S3DNA substrate or the S1 RNA substrate, 30 mM EPPS at pH 7.5, 50 MM MgCl₂and 2 mM spermidine were admixed to form endodeoxyribonuclease reactionadmixtures. The endodeoxyribonuclease reaction admixture was maintainedat 50° C. for one hour. The cleavage of substrate by each of theseribozymes is detected as the appearance of a slower migrating ribozymecaused by the ligation of the cleaved substrate to the ribozyme (FIG. 6,Lanes 2 and 3).

[0103] Focusing on the DNA substrate S3, two rounds of selectiveamplification were performed to recover the nucleic acid enzymes, inthis case endodeoxyribonucleases, capable of cleaving DNA from acollection of ribozyme structural variants including, wild-type, DeltaP6, Delta P2, Delta P9, Delta P6/P6, and Delta P2/P9. These structuralvariants (1 μM, internally labelled with 1 μCi/nmole ³²P-GTP) wereadmixed with 2 μM DNA substrate S3, 30 mM EPPS at pH 7.5, 50 mM MgCl₂and 2 mM spermidine and maintained at 50° C. for 1 hour. The first roundof cDNA synthesis was carried out by admixing a 20-fold excess ofd(TAT₃AT₃CGAGT) primer, heating the solution to 65° C. for 5 minutes inthe presence of 50 mM Tris-HCl at pH 7.5 and 5 mM DTT and then rapidlycooling the solution to 0° C. The solution was then made to contain 6 mMMgCl₂, 100 μM (each) dNTPs and 1 U/μl of AMV reverse transcriptase. Theresulting solution was maintained at 37° C. for 20 minutes. A smallaliquot of the solution was removed and analyzed by electrophoresis in a5% polyacrylamide/8 M urea gel. After the first cDNA synthesis, theselected reverse transcriptant of the Delta P6, Delta P2, Delta P9, andDelta P2/9 ribozymes can be seen in Lane 5 of FIG. 6.

[0104] The RNA is destroyed by alkaline hydrolysis and the monomersremoved by ethanol precipitation. RNA was transcribed from the cDNA byadmixing a 20-fold excess of d(ATCGATAATACGACTCACTATAGGAGGGAAAAGTTATCAGGC) primer, heating the resulting solutionto 65° C. for 5 minutes in the presence of 50 mM Tris-HCl at pH 7.5 and5 mM DTT and then rapidly cooling the solution to 0° C. The solution isthen made to contain 15 MM MgCl₂, 2 mM spermidine, 100 μM (each) dNTPs,2 mM (each) NTPs 1 U/μL AMV reverse transcriptase, 0.2 U/μL DNApolymerase I (Klenow fragment) and 20 U/μL of T7 RNA polymerase. Thesolution was maintained at 37° C. for 1 hour to allow RNA to betranscribed from the cDNA. This step results in a large amplification ofthe ribozymes having the desired catalytic activity (FIG. 6, Lane 6,{fraction (1/50)} of the material).

[0105] A second round of cDNA synthesis was performed using an equalmolar mixture of the primers d(CGAGTACTCCAAAC) and d(CC-AGTACTCCGAC) torestore the 3′ end of the RNA. The remainder of the cDNA D synthesis wasperformed as above. The resulting reaction products were analyzed byelectrophoresis (FIG. 6, Lane 7).

[0106] A second round of RNA synthesis was performed using the remainingreaction mixture using the RNA synthesis conditions described above. Aportion representing {fraction (1/50)} of the resulting reactionproducts were analyzed by gel electrophoresis and are shown in FIG. 6,Lane 8.

[0107] This system allowed the selection of a ribozyme having a desiredcatalytic activity from a mixture of ribozymes.

[0108] The foregoing specification, including the specific embodimentsand examples, is intended to be illustrative of the present inventionand is not to be taken as limiting. Numerous other variations andmodifications can be effected without departing from the true spirit andscope of the present invention.

I claim:
 1. A method for specifically cleaving a single-stranded DNAmolecule, comprising the steps of: (a) providing a first RNA moleculethat is a group I intron that cleaves a second RNA molecule to leave a3′-OH, said first RNA molecule having a deoxyribonuclease activity; and(b) contacting said first RNA molecule with said single-stranded DNAmolecule under conditions which allow said first RNA molecule to causesaid single-stranded DNA molecule to be cleaved, said conditionsincluding providing Mg²⁺ ions and guanosine or guanosine triphosphate ata pH between about 6.0 and about 9.0 and a temperature between about150C and about 60° C.
 2. The method of claim 1, further comprisingproviding said RNA molecule in a reaction medium at a concentrationsufficient to cause cleavage of at least 1% of a population of the DNAmolecules in an hour.
 3. The method of claim 1, further comprisingproviding said RNA molecule in a reaction medium at a concentrationsufficient to cause cleavage of at least 10% of a population of the DNAmolecules in an hour.
 4. The method of claim 1, wherein said RNAmolecule comprises the portions of an RNA molecule of Tetrahymena havingsaid deoxyribonuclease activity.
 5. The method of claim 4, wherein saidRNA molecule is L-19, L-21, or an RNA molecule comprising the portionsof L-19 having said deoxyribonuclease activity.
 6. The method of claim1, wherein said RNA molecule comprises a binding site forsingle-stranded DNA, which binding site is complementary to nucleotidesadjacent to a cleavage site on said single-stranded DNA molecule.